Bioethanol is one of the most promising alternatives to petroleum-based fuels. Lignocellulosic biomass, such as woods and agricultural residues, is an attractive feedstock for bioethanol production because of their relatively low cost, great abundance, sustainable supply and without food conflict. Lignocellulosic materials are mainly composed of cellulose, hemicellulose, and lignin. Of these, only cellulose and hemicellulose can be used to produce ethanol by fermentation of monomeric sugars obtained by saccharification including chemical or enzymatic hydrolysis. The cellulose fraction is made up of glucose, which is the most abundant sugar in lignocellulosic biomass, while 20-40% of the biomass is hemicellulose, which consists mostly of xylose. Literature often discloses using Pichia sp. as a strain for xylose fermentation because of its high performance in turning xylose into ethanol. However, Pichia sp. is intolerant to a high ethanol concentration and environmental inhibitors. Therefore, this has resulted in this strain with poor industrial application. Normally, in the pretreatment process of cellulosic raw materials, fermentation inhibitors, such as acetic acid, furfural, and hydroxymethyl furfural, each having a range of concentration levels, are produced, depending on reaction conditions. For example, 0.5˜2.0 g/L of furfural reduces productivity by 29˜95% and growth by 25˜99%, and 1.0-5.0 g/L of hydroxymethyl furfural reduces productivity by 17˜91% and growth by 5˜99%. Hence, lignocellulosic hydrolysates produced in the pretreatment process is usually undergone an overliming process to remove furfural, so as to detoxify the fermentation inhibitors produced in the pretreatment process and thereby ensure the success of the hydrolysate fermentation process. However, the overliming process not only causes a loss of xylose but also contributes to the production of gypsum sludge; hence, the processing and disposal of the resultant gypsum sludge incurs costs and equipment, thereby increasing production costs.
Saccharomyces cerevisiae is the most attractive ethanol-producing microorganism because of its high ethanol productivity, high inhibitory compounds tolerance and safety as a GRAS organism. However, wild type S. cerevisiae strains rapidly ferment glucose, mannose and galactose, but not xylose. Thus, to achieve economically feasible ethanol fermentation, genetically engineered S. cerevisiae has been developed to improve the capacity for converting xylose into ethanol. A number of metabolic engineering strategies to enhance ethanolic xylose fermentation in S. cerevisiae have been explored. Several approaches have been prospected to express a xylose utilization pathway from naturally pentose-utilizing bacteria and fungi in S. cerevisiae either by introducing genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), or by introducing the gene encoding xylose isomerase (XI). Pichia stipitis, a naturally pentose-utilizing fungus, has been chosen as the source of the heterologously expressed enzymes because of its high ethanol yield from xylose, despite only under oxygen limitation. However, the S. cerevisiae strains expressing the XR and XDH from P. stipitis produced xylitol, and the ethanol yield from xylose is low. This is attributed to the cofactor imbalance between XR and XDH. Heterologous expression of bacterial XI genes in S. cerevisiae has been tried for many years. However, less actively expressed XI has been reported. Despite the relatively high activity of Piromyces XI in S. cerevisiae, the expression of this enzyme only enables this strain to grow slowly on xylose, suggesting that the xylose metabolic flux in S. cerevisiae is not only affected by the conversion of xylose to xylulose.
The flux of metabolism from xylose to ethanol is affected at several levels in the pathway. The transport of xylose in S. cerevisiae occurs through non-specific hexose transporters, but the affinity of xylose is one to two orders of magnitude lower than hexose sugars. Therefore, xylose transport is early considered a rate-controlling step for ethanolic xylose fermentation. In addition, the production of xylitol during xylose consumption by recombinant xylose-utilizing S. cerevisiae is ascribed to the difference in cofactor preferences between the enzymes in the initial xylose utilization pathways. Xylitol formation in recombinant S. cerevisiae has been reduced by expressing mutated XR or XDH with altered cofactor affinity or to increase the NADPH pool by overexpressing the heterologous GADPH enzyme. The fact is that not only the cofactor preferences of the enzymes are involved, but also the levels of the XR and XDH activities affect xylitol formation during xylose fermentation. Increase of the XR and XDH activity, allowing an increased flux in the initial xylose pathway, significantly reduces xylitol accumulation. Increases of the XR and XDH activities have been observed in mutant S. cerevisiae strains with improved xylose utilization. Similarly, high activity of Piromyces XI allows higher xylose fermentation rates than the lower bacterial XI activity. The S. cerevisiae genome contains the gene XKS1 coding for XK, but the XK activity in wild-type S. cerevisiae is too low to support ethanolic xylose fermentation in strains engineered with a xylose metabolic pathway. However, it is only when additional copies of XKS1 are expressed that recombinant xylose utilizing S. cerevisiae produces ethanol from xylose. But, unregulated kinase activity may cause a metabolic disorder. It has experimentally been shown that only fine-tuned expression of XKS1 in S. cerevisiae has improved ethanol fermentation from xylose. Above all, there is not just one rate-limiting step in metabolic flux from xylose to ethanol by S. cerevisiae and therefore, strain engineering for enhanced capacity for xylose fermentation remains a challenge.